1. Field of the Invention
This invention relates to 1-benzylimidazole derivatives, and more specifically, to the use of such compounds as pharmaceutical agents, e.g., as modulators of blood glucose levels. This invention also relates to pharmaceutical compositions comprising such compounds and to the use of such compounds in treating a variety of disorders associated with feeding and food metabolism. Additionally, this invention relates to the use such compounds as probes for the localization of cellular receptors that are involved in the modulation of blood glucose levels.
2. Background
Diabetes mellitus is a chronic syndrome of impaired carbohydrate and fat metabolism resulting from insufficient insulin secretion and/or target tissue insulin resistance. It occurs in two major forms: insulin-dependent diabetes mellitus (IDDM, Type 1) and non-insulin-dependent diabetes mellitus (NIDDM, Type 2). These forms differ in their etiology, age of onset and treatment. Type 1 is often characterized by onset during childhood and the patients typically become fully dependent upon exogenous insulin to sustain life. The disorder is associated with a lack of insulin production by the pancreatic Islets of Langerhans. The disease is generally marked by a drastic reduction in the number of insulin secreting islet beta cells.
NIDDM usually appears later in life (age 40-60) and is often associated with obesity. Patients with NIDDM show normal basal levels of insulin but display an abnormal insulin secretion response (delayed or reduced) to a glucose load. As the disease progresses, insulin target tissues show signs of diminished response to insulin (insulin resistance). Effective treatment of the disorder is usually obtained by dietary control, with or without the use of oral hypoglycemic drugs. Sulphonylureas are a class of hypoglycemic compounds used in the treatment of NIDDM. These drugs exert their action by causing insulin to be released from intracellular stores. Care must be taken in the administration of these agents in order to not induce severe hypoglycemia due to excessive insulin release. In addition, overdose may deplete insulin stores to a point requiring administration of exogenous insulin.
The discovery that glucose administered via the gastrointestinal tract provides greater stimulation of insulin release than a comparable glucose challenge given intravenously led to the identification of certain gut secreted `incretin` hormones which augment glucose stimulated insulin secretion, and the identification of specific cell surface receptors that modulate the effects of such incretin hormones. Glucagon-like Peptide-1 (7-36)-amide (GLP-1) is one such incretin hormone that is secreted from gastrointestinal L cells in response to food intake and increases insulin secretion from pancreatic beta cells (Fehmann, H. C.; Goke, R. and Goke, B. (1995) Endocr. Rev. 16: 390-410). GLP-1 exerts its actions via binding to a G-protein-linked receptor expressed in islet .beta.-cells.
Unlike the sulphonylureas, the effects of GLP-1 are dependent upon plasma glucose concentration in that the insulinotropic effects of GLP-1 are abolished at low plasma glucose levels. In addition to its stimulation of insulin secretion, GLP-1 also increases insulin synthesis (Drucker, D. J. (1987) Proc. Natl Acad. Sci USA 84: 3434-3438), inhibits glucagon secretion (Kawai (1989) Endocrinology 124: 1768-1773) and delays gastric emptying (Nauck, Md. (1995) Gut 37(2): A124). This combination of actions gives GLP-1 unique potential therapeutic advantages over other agents presently used to treat non-insulin dependent diabetes mellitus. In a clinical trial of patients with NIDDM it was found that subcutaneous administration of GLP-1 could normalize postprandial glucose levels (Todd et al. (1997) Eur. J. Clin. Invest. 27: 533-536). Drugs that mimic the action of GLP-1, i.e. stimulate insulin secretion from pancreatic .beta.-cells, but only at higher than normal blood gluose levels, are particularly desirable for us in the treatment of NIDDM. Such drugs may work by modulating the signal-transducing activity of the GLP-1 receptor.
In clinical studies GLP-1 has been shown to reduce appetite and increase satiety in both normal weight and obese subjects (see, e.g., Christophe J. Ann. N Y Acad. Sci. (1998) 865:323-335 and Gutwiller, J. P., Am. J. Physio. (1999) 276: R1541-1544). Thus drugs that modulate the activity of the GLP-1 receptor may be useful for the treatment of obesity and eating disorders.
The effect of a compound on blood glucose levels can be determined in vivo, through the use a glucose tolerance test, in which the blood glucose levels laboratory animals subjected to a glucose challenge are monitored in the presence and absence of the compound. The effects of test compounds on glucose tolerance may be evaluated in non-diabetic laboratory animals as discussed in Wang et al., J. Clin. Invest. (1995) 95: 417-421 and Holst, Curr. Opinion in Endocrinology and Diabetes (1998) 5: 108-115.
Alternatively, the effects of test compounds on blood glucose levels may be assessed in an animal model of diabetes, e.g., streptozotocin (STZ)-induced diabetes. Such assays have been disclosed by Tancrede et al. (Br. J. Exp. Path. (1983) 64: 117-123), Junod et al. (J. Clin. Inv. (1969) 48: 2129-2139, Rondu et al. (J. Med. Chem. (1997) 40:3793-3803), and Maloff and Boyd (Diabetologia (1986) 29: 295-300).
In vitro experiments that monitor the interaction of the compound with GLP-1 receptors may also be used to reliably predict the effects of a compound on blood glucose levels. In one such experiment the interaction of compounds with GLP-1 receptors, expressed either recombinantly or naturally in high abundance in certain cell lines, may be determined by a cell-based luciferase screen or by binding experiments measuring competition binding e.g., with a labeled GLP-1 ligand such as GLP-1 or GLP(7-36) peptide.
Receptors that are coupled to the G.sub.s stimulatory G-protein subunit transduce intracellular signals via the adenylate cyclase pathway. Stimulation of these receptors with an agonist typically results in an elevation of cytoplasmic cAMP levels which can trigger the subsequent transcription of a variety of genes, generally those with promoters containing binding sites (cAMP responsive elements--CREs) for the transcription factor, CREB (CRE binding protein).
Receptor modulation may be measured via measurement of transcriptional activation of a firefly luciferase reporter gene. Such an assay may use a Chinese hamster ovary cell line (CHO-K1) stably transfected with a GLP-1 receptor (a G.sub.s coupled receptor) expression plasmid and a luciferase reporter plasmid, wherein luciferase expression is under the transcriptional control of multiple CREs. In these cell lines, the GLP1 agonist GLP(7-36) peptide stimulates luciferase expression in a dose dependent manner with a potency (EC.sub.50.about.20 pM) similar to the data reported by Gromada et al. (1995) FEBS Lett. 373: 182-186.
Compounds are screened by seeding 15,000 cells per well in opaque multi-well plates. Cells are then incubated overnight in a tissue culture incubator. Compounds are dispensed to a final concentration of 4 uM in 1% DMSO. After 6 hours of incubation, cells are assayed for luciferase activity, which is measured in a luminometer.